The Odd "RB" Phage-Identification of Arabinosylation as a New Epigenetic Modification of DNA in T4-Like Phage RB69

Abstract

In bacteriophages related to T4, hydroxymethylcytosine (hmC) is incorporated into the genomic DNA during DNA replication and is then further modified to glucosyl-hmC by phage-encoded glucosyltransferases. Previous studies have shown that RB69 shares a core set of genes with T4 and relatives. However, unlike the other “RB” phages, RB69 is unable to recombine its DNA with T4 or with the other “RB” isolates. In addition, despite having homologs to the T4 enzymes used to synthesize hmC, RB69 has no identified homolog to known glucosyltransferase genes. In this study we sought to understand the basis for RB69’s behavior using high-pH anion exchange chromatography (HPAEC) and mass spectrometry. Our analyses identified a novel phage epigenetic DNA sugar modification in RB69 DNA, which we have designated arabinosyl-hmC (ara-hmC). We sought a putative glucosyltranserase responsible for this novel modification and determined that RB69 also has a novel transferase gene, ORF003c, that is likely responsible for the arabinosyl-specific modification. We propose that ara-hmC was responsible for RB69 being unable to participate in genetic exchange with other hmC-containing T-even phages, and for its described incipient speciation. The RB69 ara-hmC also likely protects its DNA from some anti-phage type-IV restriction endonucleases. Several T4-related phages, such as E. coli phage JS09 and Shigella phage Shf125875 have homologs to RB69 ORF003c, suggesting the ara-hmC modification may be relatively common in T4-related phages, highlighting the importance of further work to understand the role of this modification and the biochemical pathway responsible for its production.

Keywords: RB69; T4 phage; anion exchange chromatography; arabinosyl-hmC (ara-hmC); epigenetic modification; glucosyl-hmC (ghmC); glucosyltransferase transferase; hydroxymethylcytosine (hmC); matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry; restriction.

Figure 1

Monosaccharide composition analysis by high-pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). (A) monosaccharide standards; (B) hydrolysate of DDY1 sample; (C), hydrolysate of T4; and (D) hydrolysate of RB69 DNA sample. Abbreviations used: GalN, galactosamine; GlcN, glucosamine; Gal, galactose; Glc, glucose; Man, mannose.

Figure 2

High-pH anion exchange chromatography of monosaccharide standards and RB69 DNA hydrolysate. HPAEC chromatograms of (A,B) monosaccharide standards and (C,D) co-injection of monosaccharides with RB69 DNA hydrolysate. Abbreviations used: GalN, galactosamine; GlcN, glucosamine; Gal, galactose; Glc, glucose; Man, mannose; *, Unidentified sugar; l-rha, l-rhamnose; d-Ara, d-arabinose; d-Xyl, d-xylose; d-Rib, d-ribose.

Figure 3

High-pH anion exchange chromatography of arabinose standard and RB69 DNA. HPAEC chromatograms of (A) arabinose standard, (B) RB69 DNA hydrolysate, and (C) co-injection of arabinose standard and sample RB69 DNA hydrolysate.

Figure 3

High-pH anion exchange chromatography of arabinose standard and RB69 DNA. HPAEC chromatograms of (A) arabinose standard, (B) RB69 DNA hydrolysate, and (C) co-injection of arabinose standard and sample RB69 DNA hydrolysate.

Figure 4

Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry mass spectrometry analysis of peracetylated samples of T4 and RB69 DNA hydrolysates. (A) Structure of glucose peracetate; (B) Structure of arabinose peracetate and mass spectrums of (C) peracetylated glucose, (D) peracetylated arabinose, (E) peracetylated T4 DNA hydrolysate, and (F) peracetylated RB69 DNA hydrolysate.

Figure 5

Phage RB69 arabinosyltransferase candidate ORF003c. (A) Region of the RB69 genome encoding the putative arabinosyltransferase gene ORF003c. Genes with homologs in T4 are shaded light brown and genes unique to RB69 are shaded dark blue (B) Structure of the T4 a-gt (PDB 1XV5) and (C) predicted structure of RB69 ORF003c by Phyre2 (intensive mode).

Figure 6

Candidate RB69 genes involved in the synthesis of ara-hmC DNA. (A) Region of the RB69 genome encoding novel genes relative to T4. RB69 genes with homologs in T4 are shaded light brown. RB69 genes with no homolog in T4 are shaded dark blue. (B) Scheme summarizing the similarity between RB69 ORF53_52c and ORF055c with Butyrivibrio proteoclasticus and E. coli proteins.